Background: Acute lung injury (ALI) is a severe condition distinguished by inflammation and impaired gas exchange in the lungs. Staphylococcus Aureus, a common bacterium, can cause ALI through its virulence factors. Aloe vera is a medicinal plant that has been traditionally used to treat a variety of illnesses due to its anti-inflammatory properties. Chitosan nanoparticles is biocompatible and totally biodegradable material have shown potential in drug delivery systems. Aim: To explore antibacterial activity of Aloe vera-loaded chitosan nanoparticles (AV-CS-NPs) against S. aureus in vitro and in vivo with advanced techniques. Methods: The antibacterial efficacy of AV-CS-NPs was evaluated through a broth microdilution assay. Additionally, the impact of AV-CS-NPs on Staphylococcus aureus (S. aureus)-induced ALI in rats was examined by analyzing the expression of genes linked to inflammation, oxidative stress, and apoptosis. Furthermore, rat lung tissue was scanned histologically. The rats were divided into three groups: control, ALI, and treatment with AV-CS-NPs. Results: The AV-CS-NPs that were prepared exhibited clustered semispherical and spherical forms, having an average particle size of approximately 60 nm. These nanoparticles displayed a diverse structure with an uneven distribution of particle sizes. The maximum entrapment efficiency of 95.5%±1.25 was achieved. The obtained findings revealed that The MIC and MBC values were determined to be 5 and 10 ug/mL, respectively, indicating the potent bactericidal effect of the nanoparticles. Also, S. aureus infected rats explored upregulation in the mRNA expression of TLR2 and TLR4 compared to healthy control groups. AV-CS-NPs treatment reverses the case where there was repression in mRNA expression of TLR2 and TLR4 compared to S. aureus-treated rats. Conclusion: These nanoparticles can serve as potential candidates for the development of alternative antimicrobial agents.